BmPGRP-L4 drives antibacterial activity and prophenoloxidase pathway activation in the silkworm Bombyx mori.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors that play critical roles in detecting bacterial infections and activating immune responses in insects. Although twelve PGRPs have been identified in the silkworm Bombyx mori, the functions of most long-type PGRPs, including BmPGRP-L4, remain largely unexplored. Previously, we reported that BmPGRP-L4 is highly expressed in the midgut and can be induced by bacterial challenge, and that it functions as a negative regulator of the humoral immune response. In this study, we produced recombinant BmPGRP-L4 protein and systematically investigated its biochemical and immunological functions. We demonstrated that BmPGRP-L4 binds to both Lys-type peptidoglycans from Staphylococcus aureus and DAP-type peptidoglycans from Escherichia coli, indicating its recognition capability for both Gram-positive and Gram-negative bacteria. Enzyme activity assays confirmed that BmPGRP-L4 possesses active amidase activity that is enhanced by zinc ions. Furthermore, we showed that BmPGRP-L4 exhibits antibacterial activity against both S. aureus and E. coli, and induces bacterial agglutination. Importantly, we revealed that BmPGRP-L4 is involved in the prophenoloxidase activation pathway, serving as a receptor that triggers melanization upon recognition of bacterial peptidoglycans. Collectively, these findings establish BmPGRP-L4 as a multifunctional immune effector in the silkworm, playing dual roles as a pattern recognition receptor for the prophenoloxidase pathway and as an antibacterial protein, which provides a molecular basis for its regulatory function in humoral immune responses.